The present invention relates to certain novel pyridine compounds. The compounds of the present invention are especially useful for producing new acyl derivatives of 4-demethylpenclomedine (also referred to herein as DM PEN). The acyl derivatives, in turn, exhibit anticancer activity, and are the subject of U.S. patent application Ser. No. 60/116,675 filed Jan. 21, 1999 and entitled xe2x80x9cAcyl Derivatives of 4-Demethylpenclomedine, Use and Preparation Thereofxe2x80x9d, the entire disclosure of which is incorporated herein by reference. Certain of the pyridine compounds of the present invention also exhibit anticancer activity. Accordingly, the present invention also relates to pharmaceutical compositions comprising those particular pyridine compounds, as well as a method of using them in treating cancer in a mammal. The present invention also relates to a method for producing the compounds of the present invention and to a method for producing the acyl derivatives of 4-demethylpenclomedine from the pyridine compounds of the present invention.
Even though significant advances have occurred in treatment of cancer, it still remains a major health concern. It has been reported that cancer is the cause of death of up to one of every four Americans.
Included among the known chemotherapeutic drugs are carmustine, doxorubicin, methotrexate, TAXOL(copyright), nitrogen mustard, procarbazine, and vinblastine, to name only a few. However, many chemotherapeutic drugs also produce undesirable side effects in the patient. For example, U.S. Pat. No. 4,717,726 reportedly discloses a compound suitable for inhibiting the growth of certain types of malignant neoplasms in mammals. See also Plowman et al., Cancer Res., 49 (1989), 1909-1915. The disclosed compound, 3,5-dichloro-2,4-dimethoxy-6-(trichloromethyl)pyridine, also known as penclomedine, is not satisfactory as a chemotherapeutic, however, because it is known to produce certain undesirable side effects especially in the central nervous system.
For example, neurological and hematological toxicities of penclomedine have been reported in preclinical and early clinical studies. Dose related neurotoxicity, consisting of muscle tremors, incoordination, convulsions and reduced activity, has been observed in rats. Neurotoxicity appears to be related to peak plasma drug concentrations, as it developed during or immediately after infusion and could be ameliorated by decreasing the rate of drug administration. In dogs, severe emesis and seizures have been associated with plasma penclomedine levels above 30 xcexcM. Neurotoxicity, consisting of dysmetria, ataxia, and vertigo, was also the principal dose limiting toxicity of penclomedine administered as a one hour infusion for 5 consecutive days in patients with advanced solid tumors. The presence of these toxicities, at much lower peak plasma concentrations compared to those reported in preclinical studies, may preclude the administration of higher doses of penclomedine and the achievement of concentrations associated with optimal antitumor activity. Berlin et al., Proc. Amer. Assoc. Cancer Res., 36, 238 (1005); O""Reilly et al., Proc. Amer. Soc. Clin. Oncol., 14, 471 (1995).
Some relevant background art can be found in O""Reilley et al., Clinical Cancer Research, 2 (March 1996), 541-548. This reference describes a study to assess the distribution of 14C-penclomedine in the tissues and tumors of tumor-bearing rats. The study found that the predominant radioactive species in the brain was penclomedine, which may explain the observed neurotoxicity of the drug.
More recently, 3,5-dichloro-2-methoxy-4-hydroxy-6-(trichloromethyl)pyridine or 4-demethylpenclomedine has been suggested as a compound for treating cancer. See WO 97/46531 to Hartman et al.
Notwithstanding the advances in cancer treatment that have been made, there still remains room for improved drugs that are effective in treating cancer, while at the same time exhibit reduced adverse side effects.
The present invention relates to new pyridine compounds that find especial utility as precursors for producing certain acyl derivatives of 4-demethylpenclomedine which, in turn, possess anticancer activity as will be discussed hereinbelow.
The compounds of the present invention are represented by the following formulae: 
wherein
each R1 is independently an alkyl group provided that at least one R1 is an alkyl group containing at least two carbon atoms; and
each R2 is independently H, acyl or an alkyl group having at least two carbon atoms or R2 can be methyl in position 4 provided that R2 is acyl in position 6.
The present invention also relates to a process for preparing compounds of formula I by reacting 3,4,5,6-tetrachloro-2-(trichloromethyl)pyridine with an alcohol in the presence of a base to produce the corresponding 3,5-dichloro-4,6-dialkoxy-2-(trichloromethyl)pyridine of formula I.
The compound according to formula II wherein both R2 are H can be prepared by reacting the compound of formula I with a dealkylating agent such as anhydrous aluminum chloride to produce didemethylpenclomedine.
The compounds according to formula II wherein at least one of R2 is acyl can be prepared by reacting didemethylpenclomedine with an acylating agent to form a corresponding acyl compound of formula II.
The acyl compound of formula II can then be reacted with an alkylating agent such as (trimethylsilyl)diazomethane or diazomethane to produce the acyl derivatives of 4-demethylpenclomedine.
These novel acyl derivatives of 4-demethylpenclomedine compounds are represented by the formula: 
Pharmaceutically acceptable salts thereof can also be provided.
The present invention also relates to compounds represented by the formula 
wherein one of R3 or R4 is Cl and the other of R3 or R4 is O-alkyl.
A further aspect of the present invention is concerned with producing compounds of formula IV. The compounds of formula IV can be produced by reacting 3,4,5,6-tetrachloro-2-(trichloromethyl)pyridine with an alcohol in the presence of a base and separating the desired compound from the reaction mixture.
Another aspect of the present invention relates to an alternative method for producing a compound of formula I above by reacting a compound of the formula IV 
wherein one of R3 or R4 is Cl and the other of R3 or R4 is O-alkyl with an alcohol in the presence of a base.
In addition, compounds of formula I above can be produced by reacting a compound of the formula 
wherein R1 is an alkyl group having at least two carbon atoms with an alkylating agent.
A still further aspect of the present invention is directed to producing a compound of formula II above wherein R2 in position 4 is H and R2 in position 6 is an alkyl which comprises reacting a compound represented by the formula I 
wherein each R1 is independently an alkyl group with R1 in position 4 having at least two carbon atoms with a dealkylating agent to selectively dealkylate said compound in position 4.
Compounds of the present invention wherein the R1 group located at position 4 is ethyl show anticancer activity. Accordingly, the present invention also relates to pharmaceutical compositions containing these compounds wherein the R1 group located at position 4 is ethyl, and to methods of using such compounds in treating cancer in a mammal.
Still other objects and advantages of the present invention will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described only the preferred embodiments of the invention, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.
The present invention is concerned with pyridine compounds represented by the following formulae: 
wherein
each R1 is independently an alkyl group provided that at least one R1 is an alkyl group containing at least two carbon atoms; and
each R2 is independently H, acyl or an alkyl group having at least two carbon atoms or R2 can be methyl in position 4 provided that R2 is acyl in position 6.
The alkyl group typically contains 1 to 22 carbon atoms and preferably 2 to 22 carbon atoms.
The R2 acyl groups can be straight or branched chained, can be unsubstituted or substituted such as with halogen such as Cl, Br and I, and/or include 5 and 6 membered rings. The ring moiety can be a carbocycle or a heterocycle including a hetero atom such as O, S or N. Typically, the acyl group contains 1-12 carbon atoms.
The present invention is also directed to compounds represented by the formula 
wherein one of R3 or R4 is Cl and the other of R3 or R4 is O-alkyl.
The alkyl group typically contains 1 to 22 carbon atoms. Preferably R3 is O-alkyl and R4 is Cl.
The compounds of formula IV can be produced by reacting 3,4,5,6-tetrachloro-2-(trichloromethyl)pyridine with an alcohol in the presence of a base and separating said compound from the reaction mixture. A typical base is NaOH. This process is preferably carried out under reflux with a typical alcohol being methanol.
Although many of the above precursor compounds do not exhibit any appreciable anti-cancer activity, it has been observed that at least a few of the precursor compounds show active anti-cancer activity. These compounds have the structure wherein at least the R1 group located at position 4 is ethyl and include 3,5-dichloro-4,6-diethoxy-2-(trichloromethyl)pyridine and 3,5-dichloro-4-ethoxy-6-methoxy-2-(trichloromethyl)pyridine. Therefore, those compounds wherein at least the R, group at position 4 is ethyl or pharmaceutically acceptable salts thereof can be used in treating cancer in a mammal. However, these compounds are not as active as the acyl derivatives of 4-demethylpenclomedine according to copending U.S. patent application Ser. No. 60/116,675 filed Jan. 21, 1999 and entitled Acyl Derivatives of 4-Demethylpenclomedine, Use and Preparation Thereof, the entire disclosure of which is incorporated herein by reference, and also exhibit higher toxicity as compared to these acyl derivatives of DM-PEN.
These novel acyl derivatives of 4-demethylpenclomedine compounds are represented by the formula: 
and pharmaceutically acceptable salts thereof.
The acyl groups can be straight or branched chained, can be unsubstituted or substituted such as with halogen such as Cl, Br and I, and/or include 5 and 6 membered rings. The ring moiety can be a carbocycle or a heterocycle including a hetero atom such as O, S or N. Typically, the acyl group contains 1-12 carbon atoms.
Examples of such suitable acyl groups are the following, each of which has been evaluated for activity according to the present invention, and each has been characterized by mass spectroscopy: 
Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulfonic, for example p-toluenesulfonic acid.
It has been found that the acyl compounds of formula III are surprisingly and advantageously useful in treating mammalian cancer, especially human cancer. These compounds have been shown to exhibit generally superior activity in comparison to 4-demethylpenclomedine and penclomedine. Moreover, these are believed to possess reduced toxicity in comparison to both demethylpenclomedine and penclomedine (PEN).
It is further noted, as will be discussed below, that the acyl compounds of formula III are not considered to be prodrug forms of DM-PEN. Both penclomedine (PEN) and DM-PEN are inactive as cytotoxic agents in vitro but must be metabolized to produce cytotoxicity, as demonstrated by their anticancer activity in vivo, which indicates that DM-PEN, as well as PEN, is a prodrug of the ultimate activated metabolite. The proposed mechanism by which PEN and DM-PEN exhibit cytotoxicity in vivo is shown in Scheme 1 and indicates that PEN and DM-PEN are on the same metabolic activation pathway, which includes a non-acylated, free radical activated for DNA crosslinking. In contrast, it is believed (Scheme 2) that the acyl derivatives (DM-ACYL-PEN) are converted via the liver to an acylated free radical (rather than a non-acylated free radical) and, as such, are not fully activated for DNA crosslinking but remain in a prodrug form as they exit the liver and enter the circulation. The partially activated acylated free radical is transported to cells via circulation and forms an adduct with nuclear DNA. Subsequently, the adduct is deacylated by general cellular esterases particularly in the tumor cell, which allows for in situ, full activation and possibly reduced generalized host toxicity in comparison to PEN and DM-PEN, both of which are fully activated in the liver for DNA adduction and crosslinking before being transported via circulation to tumor and non-tumor cells. Deacylation generates a 4-hydroxy moiety on the pyridine nucleus, which then tautomerizes via the enol-keto mechanism to produce an alpha-haloketo moiety at the 4,5-positions in which the 5-chloro function is activated for displacement by a nucleophilic moiety on the DNA for completion of a DNA crosslink, as shown in Scheme 2. This mechanism is further supported by the generally superior activity of the acyl derivatives in comparison to DM-PEN vs. both subcutaneously (s.c.)- and intracerebrally (i.c.)-implanted human MX-1 mammary tumor xenograft, s.c.-implanted human U251 CNS tumor xenograft, and both parent, and particularly, cyclophosphamide (CPA)-resistant lines of P388 leukemia, as shown in Tables 1-4 below. 
The acyl derivatives of DM-PEN can be synthesized by the following route that employs the pyridine compounds of the present invention. 
As illustrated above, the acyl derivatives of DM-PEN can be produced by reacting 3,4,5,6-tetrachloro-2-(trichloromethyl)pyridine with an alcohol in the presence of a base to produce the corresponding 3,5-dichloro-4,6-dialkoxy-2-(trichloromethyl)pyridine (i.e. compound of formula I); which in turn is reacted with a dealkylating agent such as anhydrous aluminum chloride to produce didemethylpenclomedine. (Compound of formula II wherein each R2 is H.)
Of course, other dealkylating agents can be used if desired. The didemethylpenclomedine is then reacted with an acylating agent to form the corresponding acyl derivatives of demethylpenclomedine (i.e. compound of formula II wherein at least one R2 is acyl), which is then reacted with an alkylating agent such as (trimethylsilyl)diazomethane to produce the acyl derivatives of DM-PEN.
It is understood that other acylating agents and alkylating agents can be used. Moreover, various reaction parameters such as temperature, relative amounts, and pressure can be selected by those skilled in the art once aware of the present disclosure.
In addition, compounds of formula I disclosed above can be produced by reacting a compound of the formula 
wherein one of R3 and R4 is Cl and the other of R3 and R4 is O-alkyl with an alcohol in the presence of a base.
A typical base is NaOH, and the reaction is typically carried out under reflux.
Moreover, compounds of formula I disclosed above can be produced by reacting a compound of the formula 
wherein R1 is an alkyl group having at least two carbon atoms with an alkylating agent such as (trimethylsilyl)diazomethane.
R1 typically has 2-22 carbon atoms.
Compounds of formula II disclosed above wherein R2 in position 4 is H and R2 in position 6 is an alkyl can also be prepared by reacting a compound represented by the formula 
wherein each R1 is independently an alkyl group with R1 in position 4 having at least two carbon atoms with a dealkylating agent to selectively dealkylate said compound in position 4.
A typical dealkylating agent is dimethyl sulfoxide.
The reaction is typically carried out under elevated temperatures such as about 140 to about 160xc2x0 C., an example of which is about 150xc2x0 C.
The alkyl group in the 4 position typically contains 2-22 carbon atoms, and preferably 2-6 carbon atoms, examples of which are ethyl and n-butyl. The alkyl group is preferably straight chained. For instance under the conditions employed in Example 3 below, using isopropyl did not achieve the desired result.
The above process is suitable for producing 4-demethylpenclomedine which in turn can be used to produce the above disclosed acyl derivatives of 4-demethylpenclomedine.
Novel precursors for producing the acyl derivatives of the present invention are represented by the following formulae: 
wherein
each R1 is independently an alkyl group provided that at least one R1 is an alkyl group containing at least two carbon atoms; and
each R2 is independently H, acyl or alkyl having at least two carbon atoms or R2 can be methyl in position 4 provided that R2 is acyl in position 6.
The alkyl group typically contains 1 to 22 carbon atoms and preferably 2 to 22 carbon atoms.
The R2 acyl groups can be straight or branched chained, can be unsubstituted or substituted such as with halogen such as Cl, Br and I, and/or include 5 and 6 membered rings. The ring moiety can be a carbocycle or a heterocycle including a hetero atom such as O, S or N. Typically, the acyl group contains 1-12 carbon atoms.
Although many of the above precursor compounds do not exhibit any appreciable anti-cancer activity, it has been observed that at least a few of the precursor compounds show active anti-cancer activity. These compounds have the structure wherein at least the R1 group located at position 4 is ethyl and include 3,5-dichloro-4,6-diethoxy-2-(trichloromethyl)pyridine and 3,5-dichloro-4-ethoxy-6-methoxy-2-(trichloromethyl)pyridine. Therefore, those compounds wherein at least the R, group at position 4 is ethyl or pharmaceutically acceptable salts thereof can be used in treating cancer in a mammal. However, these precursor compounds are not as active as the acyl compounds of the present invention and also exhibit higher toxicity as compared to acyl compounds of the present invention.